Gene silencing or cosuppression by homologous transgenes introduced into the genome of plants has raised considerable interest. A transgene can inactivate the normal (endogenous) gene or another transgene of the same type in different genomic locations via a variety of mechanisms (Baulcombe, D. C. et al., Curr. Opin. Biotech. 7:173-180 (1996)). These phenomena have previously been observed in higher plants (Matzke, M. A. et al., Plant Physiol. 107: 679-685 (1995)), and related processes involved in the silencing of duplicated genes have been observed in fungi (Cogoni, C. et al., EMBO J. 15:3153-3163 (1996); Meyer, P., Biol. Chem. 377: 87-95 (1996)). Cosuppression, a reciprocal function involving interactions between the endogenous gene and the genome-integrated transgene, has been detected in the invertebrate insect Drosophila (Pal-Bhadra. M. et al. Cell 90:479-490 (1997)).
In genetically modified plants, transgenes that are stably maintained can be silenced. Transgenes can in addition cause the silencing of the endogenous plant genes if they are sufficiently homologous, a phenomenon known as co-suppression. Silencing occurs transcriptionally and post-transcriptionally but silencing of endogenous genes seems predominantly post-transcriptional (Stam, M. et al., Annals of Botany 79:3-12 (1997)). Various factors seem to play a role, including DNA methylation (Ingelbrecht, I. et al., Proc. Natl. Acad. Sci. USA 91: 10502-10506 (1994)), transgene copy number and the repetitiveness of the transgene insert (Meyer, P., Biol. Chem. 377: 87-95 (1996)), transgene expression level (Vaucheret, H. et al., Plant Cell 9:1495-1504 (1997)), possible production of aberrant RNAs (Metzlaff, M. et al., Cell 88:845-854 (1998)), and ectopic DNA—DNA interactions (Baulcombe, D. C. et al., Curr. Opin. Biotech. 7:173-180 (1996)).
An array of cis-acting DNA elements and trans-acting factors are involved in regulation of expression of pro-collagen genes, including α1(I). DNA transfection experiments have shown that two blocks of both positive and negative regulatory elements, located in the 5′-flanking region and the first intron, contribute to the transcriptional regulation of the pro-α1(I) collagen gene (Brenner, D. A. et al., Nucleic Acids Res. 17:6055-6064 (1989); Rippe, R. A. et al., Mol. Cell. Biol. 9:2224-2227 (1989)). In NIH3T3 mouse fibroblasts, which synthesize large amounts of collagen (2.2% of total protein), about 220 bp of the mouse pro-α1(I) collagen promoter carried on the construct ColCAT3 (also called pColCAT0.2) showed high transcriptional activity, comparable to that of the highly active SV40 promoter of the pSV2CAT construct. However, constructs carrying increasingly larger 5′-flanking sequences showed reduced amount of the reporter chloramphenicol acetyl transferase gene (CAT) activities of between 65% to less than 20% of that of pCOlCAT0.2 (Rippe, R. A. et al., Mol. Cell. Biol. 9:2224-2227 (1989)). The reporter gene activity being measured in these experiments was a fusion to the pro-α1(I) collagen promoter carried on the transgenic plasmid construct.
The ability to control suppression of gene expression in an animal cell will enable several practical solutions to current problems. For example, reducing expression of an oncogenic transformation effector gene, a drug resistance gene, a radioresistance gene or a viral gene, by employing an appropriate gene delivery system, could provide improved treatment for a variety of cancers and for infections by pathogens, for example, viral infections. Further, determining the effects of suppression of activity of a target gene in a cell would be a useful method for genomic analysis, for example, as a more efficient and rapidly available alternative to engineering a knock-out animal for determining the phenotypes of the cells lacking expression of the target gene. The methods of suppression and the cells thus suppressed can provide screening tools to identify drugs capable of reducing gene expression, and also to identify drugs that can reverse the suppression of gene expression.